This dissertation describes the process of optimizing a Fluorescence Lifetime
Imaging Microscopy (FLIM) system in order to observe the dynamics of enzymes in live
cancer cells. The enzyme studied throughout this research is Membrane Type 1 Matrix
Metalloproteinase (MT1-MMP) which is a membrane-bound protein principally
responsible for degrading extra-cellular matrix (ECM) proteins in the local environment
of a migrating cell. However, MT1-MMP has an intricate role in the regulation of the
cell’s migration separate from its simple proteolytic functions. In addition, the increased
expression of MT1-MMP has been positively correlated with the invasive potential of
tumor cells. In spite of the importance of MT1-MMP in understanding a cancer cell’s
decision making as it leaves a tumor, very few reports have quantitatively studied the
activity of this enzyme in live cells. Even fewer reports have examined the
spatiotemporal activity of MT1-MMP in live cells cultured in 3-dimensional settings such
as matrices of ECM proteins. These 3-dimensional settings can parallel the environment
encountered by metastasizing cells in tissues. Studying live cells in 3-dimensional
matrices is crucial for biologically relevant investigations. A cell’s morphology and
migratory behavior can vary significantly when comparisons are made between cells
cultured on two dimensional substrates and those cultured in 3-dimensional matrices. The
purpose of this project was to understand the coordinated functions of MT1-MMP as live
cancer cells interact with and move through a 3-dimensional matrix of ECM proteins.
Specifically, we are ultimately interested in the spatiotemporal activation patterns of
MT1-MMP in live cancer cells in order to build a quantitative (systems-level) model
describing MT1-MMP’s role in the cell’s decision making as it is leaves a tumor site.